Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
  • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
  • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/16—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using chemical substances
  • A61L2/18—Liquid substances
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B2/00—Preservation of foods or foodstuffs, in general
  • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
  • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
  • A23B2/729—Organic compounds; Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B2/00—Preservation of foods or foodstuffs, in general
  • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
  • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
  • A23B2/729—Organic compounds; Microorganisms; Enzymes
  • A23B2/742—Organic compounds containing oxygen
  • A23B2/75—Organic compounds containing oxygen with doubly-bound oxygen
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B2/00—Preservation of foods or foodstuffs, in general
  • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
  • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
  • A23B2/729—Organic compounds; Microorganisms; Enzymes
  • A23B2/742—Organic compounds containing oxygen
  • A23B2/754—Organic compounds containing oxygen containing carboxyl groups
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B4/00—Preservation of meat, sausages, fish or fish products
  • A23B4/12—Preserving with acids; Acid fermentation
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B4/00—Preservation of meat, sausages, fish or fish products
  • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
  • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
  • A23B4/20—Organic compounds; Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
  • A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
  • A23B7/153—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of liquids or solids
  • A23B7/154—Organic compounds; Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials

Definitions

• the present invention relates to food safety and also to the treatment of bacterial infections, in particular due to E . coli 0157 and other ' foodborne pathogenic bacteria.
• Escherichia coli 0157 is a prime example. This bacterium is the causative agent of haemorrhagic colitis and haemolytic uremic syndrome. Infections caused by E . coli 0157, though infrequent, are associated with a high level of morbidity and mortality, particularly in the young and the elderly. The severity of infections caused by E. coli 0157 and other pathogenic bacteria has attracted a high level of media attention. This has resulted in reduced public confidence in food safety, particularly in red meat products and products such as salad leaves and other vegetables which may be treated with fertilisers containing animal manure.
• Escherichia coli 0157 has a very low infectious dose and it may be carried asymptomatically by farm animals including cattle, sheep, pigs, turkeys etc. Farm animals including cattle and sheep are regarded as a primary reservoir of E. coli 0157. Moreover, this organism is repeatedly isolated from the farmyard which strongly implicates this environment in the persistence of E. coli 0157 (LeJeune et al . (2001)). Cattle faeces are a major source of contamination of meat products in slaughter houses, and slurry is a potential source of contamination of local water supplies and crops, as well as vegetables and fruit which may be eaten raw.
• feed deprivation is thought to predispose cattle to carriage of E. coli and Salmonella (Brownlie and Grau (1967) ) .
• compositions capable of reducing the numbers of, or inactivating, bacterial pathogens (including those present at any point in the food chain, or present in the environment) .
• HACCP Hazard Analysis Critical Control Points
• MRSA methicillin-resistant Staphylococcus aureus
• WO-A-99/44444 describes the use of a solution containing lactic acid and at least one ingredient chosen from hydrogen peroxide, sodium benzoate or glycerolmonolaurate at certain temperatures and durations. No mention is made of the distinction between the L- and D- isomers of lactic acid, nor is there any mention of coumarin compounds.
• Bintsis et al . (2000) show the efficacy of furocoumarins in inhibiting certain pathogens, including E. coli 0157. However, there is no mention of the combination of coumarins with lactic acid or other organic acids.
• the "organic acid” as herein defined may be any organic acid but specifically excludes the volatile fatty acids acetate, propionate and butyrate.
• the organic acid may be any organic acid but excludes short chain volatile fatty acids.
• the organic acids selected are medium chain fatty acids (eg. heptanoic and decanoic acids) , long chain fatty acids (eg.
• dodecanoic acid unsaturated acids (eg. sorbic acid) , hydroxylic acids (eg. lactic acid and citric acid), aromatic acids (eg. benzoic acid and salicyclic acid) or multicarboxylic acids (eg. citric acid and succinic acid) .
• the organic acids are hydroxylic acids such as citrate or lactate. L-isomers of optically active hydroxylic acids are preferred. It should be noted that the nomenclature for fatty acids adopted is as set out by Cherrington et al . (1991) .
• the present invention provides an anti-bacterial composition
• an anti-bacterial composition comprising an admixture of an organic acid as defined above and a coumarin or a coumarin glycoside. Lactate, citrate and benzoic acid are preferred, especially L-lactate.
• the present invention also provides a method for reducing the infective ability or inactivating bacterial pathogens by contacting said bacteria with a mixture of an organic acid as defined above and a coumarin or a coumarin glycoside.
• the method and composition of the present invention may be used to reduce the shedding of pathogens (including but not limited to E. coli 0157, Salmonella, Listeria, Campylobacter and MRSA) from animals and humans.
• pathogens including but not limited to E. coli 0157, Salmonella, Listeria, Campylobacter and MRSA
• pathogens including but not limited to E. coli 0157, Salmonella, Listeria, Campylobacter and MRSA
• the coumarins are derivatives of benzo- ⁇ -pyrone and occur in plants in the free state and as glycosides.
• the term "coumarin” will be used to describe the generic group of benzo- ⁇ -pyrone compounds, whereas “Coumarin” will refer to 1,2 benzopyrone .
• the coumarin glycosides may also be used since these are commonly converted to the free coumarin in vivo and may have the advantage of increased solubility which aids administration and absorbance .
• the sugar moiety once hydrolysed from the glycoside, can provide a sugar source for growth of beneficial bacteria.
• An example of a coumarin glycoside is esculin, which is a glycoside of esculetin.
• Furocoumarins for example psoralen, 3-methoxy-psolaren and 8-methoxy- psoralen may also be of interest.
• the composition contains from ImM to 500mM, more preferably from 20mM or 50mM to 250mM, of organic acid.
• organic acids are lactate, citrate and benzoic acid.
• the composition contains from 0.05mM to 15mM of a coumarin or a coumarin glycoside, preferably at least 0.5mM, for example 0.68mM, of a coumarin or a coumarin glycoside.
• the composition of the present invention has the advantage of being applicable to all stages of potential contamination and cross-contamination from farm to fork. Exemplary stages include use in the treatment of salad leaves and other vegetable material, in animal feeds, in water troughs on farms, in food preparation from slaughter to sale to the public, for use with plant matter intended as a foodstuff and also for treatment of animals or humans infected with said pathogens.
• the composition could be used as a disinfectant, cleaner, sterilizer for commercial and non- commercial cooling devices such as fridges and freezers.
• the composition could be used to disinfect buildings, in particular public buildings such as schools or hospitals, or to disinfect surfaces (such as floor, walls, furniture and medical devices/implements) .
• the composition could further be used in packaging material for foods (such as plastic "clingfilm” wrap) and, since the composition increases in effectiveness with increasing temperature, would protect wrapped produce taken from the refrigerated conditions in retail premises during transport .
• the composition could also find use in washing, coating or being incorporated into bandages, dressings and other coverings used to protect wounds from infection and contamination.
• composition of the present invention resides with the antioxidant properties of coumarins.
• Antioxidants have well known health enhancing and disease preventing properties by virtue in part to their ability to reduce oxidative damage to our cells which may lead to development of many clinical conditions including cancers, heart disease, Alzheimer's disease and arthritis. Accordingly, in addition to the ability of coumarins to inhibit and kill pathogens, ingestion by animals and humans will enhance the health status of the recipient and may help to prevent and treat diseases related to oxidative stress.
• the antioxidant properties of the coumarins are likely to extend the shelf-life of treated food or feed stuffs.
• composition of the present invention may be administered to animals to reduce shedding of the pathogens into the environment, for example fields, farms, water supplies, slurry and vehicles used for transportation of animals. Reduction of shedding leads to a lowering of the risk of contamination of the environment and edible and non-edible agricultural products such as fruit and vegetables and will also reduce the risk of infection to other animals and humans.
• the method of the present invention is conducted at a temperature of 37°C or lower, for example 20°C or 5°C (representing body temperature, ambient temperature and refrigerated temperature respectively) .
• a temperature of 37°C or lower for example 20°C or 5°C (representing body temperature, ambient temperature and refrigerated temperature respectively) .
• increasing the temperature increases the effectiveness of the composition.
• the numbers of Salmonella for example, are reduced by greater than 5 logio units within 60 minutes.
• a greater than 5 log ⁇ 0 unit decrease occurs in 5 minutes.
• the present invention may be administered to humans infected with pathogens so as to reduce the numbers, inhibit and kill the pathogens and consequently treat diseases caused by the infecting organism.
• the composition of the invention could be used to treat humans infected with E. coli 0157 or MRSA and thus prevent or ameliorate the effects of infection with such pathogens, which include haemorrhagic colitis and haemolytic uremic syndrome and sepicaemia.
• the composition of the present invention may further comprise a volatile fatty acid (VFA) such as, for example, acetate, butyrate or propionate.
• VFA volatile fatty acid
• the composition may comprise a polyasaccharide or other readily fermentable compound which, upon digestion in the gut, is converted to an acid such as lactate.
• composition can be used in various formats for example as sprays, liquid solutions, gels, packaging and wrapping material for foods for use on surfaces and can be delivered to the site of action in the rumen or gastro-intestinal tract by oral administration in any appropriate carrier, excipient, diluent or stabilizer.
• delivery mechanisms may be of any formulation including but not limited to solid formulations such as tablets or capsules or as feed additives; liquid solutions such as yoghurt or drinks or suspensions .
• Pathogenic E. coli strains including E. coli 0157, are of particular interest, as are Salmonella spp, Listeria spp . and Staphylococcus spp., especially MRSA.
• Figure 1 shows the susceptibility of E. coli 0157 :H7 strain NCTC 12900 to L-lactate and D-lactate.
• the L-lactate solution concentrations were 50, 100, 150 and 200mM and those of D-lactate 100, 150 and 200mM.
• the solutions all had a final pH of 3.8 and were incubated at 37°C.
• the limit of detection is 50 cfu/ml or 1.7 logio cfu/ml .
• Figure 2 illustrates the susceptibility of E. coli 0157 :H7 strain NCTC 12900 to various proportions of L-lactate and D-lactate.
• the solution concentrations were lOOmM D-lactate, 75mM D-lactate + 25mM L-lactate, 50mM D-lactate + 50mM L-lactate, 25mM D-lactate + 75mM L-lactate, and lOOmM L- lactate.
• the solutions all had a final pH of 3.8 and were incubated at 37°C.
• the limit of detection is 50 cfu/ml or 1.7 logio cfu/ml.
• Figure 3 indicates the survival of 8 E . coli 0157 :H7 strains and 8 non-0157 E.
• Figure 4 illustrates the synergy between L-lactate (50mM) and esculetin (7.5mM) in reducing the numbers of the E. coli 0157 :H7 strain NCTC 12900.
• the solutions all had a final pH of 3.8, were incubated at 37°C and the limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figure 5 illustrates the effect of temperature on the antimicrobial efficacy of 200mM L-lactate and 7.5mM esculetin on the E. coli 0157 :H7 strain NCTC 12900. Cultures were incubated at 5, 20 and 37°C. The solutions all had a final pH of 3.8 and the limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figures 6A-C depict the synergism between various coumarins (7.5mM) and L-lactate (50mM) against the E. coli 0157 :H7 strain NCTC 12900.
• the coumarins tested were scopoletin (A) , Coumarin (B) and umbelliferone (C) . Cultures were incubated at 37°C, had a final pH of 3.8 and the limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figures 7A-B illustrate the synergy between esculetin and 50mM citric acid (A) or 25mM benzoic acid in reducing the numbers of the E. coli 0157 :H7 strain NCTC 12900. The solutions all contained 7.5mM esculetin and had a final pH of 3.8. The limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figures 8A-C indicates the synergy between Coumarin and L-lactate for S . enteri tidis (A) , L- monocytogenes (B) , and an MRSA strain of S . aureus (C) .
• the solutions all contained lOmM Coumarin and had a final pH of 3.8. The limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figures 9A-B compare the effect of temperature on survival of S . enteri tidis (A) and L -monocytogenes (B) in L-lactate and Coumarin.
• the solutions contained lOmM Coumarin and 25mM L-lactate (A) or 50mM L-lactate (B) .
• the final pH was 3.8 and the limit of detection 50 cfu/ml or 1.7 logio cfu/ml.
• Figure 10 illustrates the synergy between 2% L- lactate and 6.8mM Coumarin in reducing the viability of 8 E . coli 0157 :H7 strains and 8 non-0157 :H7 E . coli strains determined over 1 hour. Results are shown as percentage survival.
• Figures 11A-D show the synergistic antimicrobial effect of 2% L-lactate and 6.8mM Coumarin on the E. coli 0157 strain NCTC 12900 (A) , S . enteri tidis (B) , L -monocytogenes (C) and S . aureus (D) .
• the limit of detection was 50 cfu/ml or 1.7 logio cfu/ml.
• Figures 12A-D illustrate the effect of temperature on the antimicrobial efficacy of 2% L-lactate and 6.8mM Coumarin for the E. coli 0157 :H7 strain NCTC 12900 (A) , S. enteri tidis (B) , L . monocytogenes (C) and S . aureus (D) .
• the limit of detection was 50 cfu/ml or 1.7 logio cfu/ml .
• Figures 13A-E demonstrate the synergistic growth inhibition of L-lactate and esculetin (A) or L- lactate and Coumarin (B-E) on E. coli 0157 :H7 strain NCTC 12900 (A-B) , S . enteri tidis (C) , L . monocytogenes (D) and S. aureus (E) .
• Table 1 shows the bacterial strains used in the examples.
• E. coli 0157 :H7 As the infectious dose of E. coli 0157 :H7 is very low, our main aim was to develop a treatment capable of killing high levels of E. coli cells.
• the E. coli strains were cultured such that there was a population of approximately 10 9 cfu/ml. The final pH for each culture was 3.8, except where 2% lactate was present in which case the final pH was around 2.0.
• Treatments consisted of various concentrations of the organic acids L-lactate, D-lactate, citrate or benzoate and the coumarins esculetin, Coumarin, scopoletin or umbelliferone . These were added to cultures which were then incubated at 5°C, 20°C or 37°C. Samples were extracted at various time intervals and the population of E. coli 0157 :H7 was determined. Other pathogens were tested in a similar manner with the exception of L . monocytogenes where the starting population was 10 8 cfu/ml.
• L-lactate final concentrations of 50, 100, 150 or 200mM
• D-lactate final concentrations of 100, 150 or 200mM
• L-lactate and D-lactate were added to prepared cultures and incubated at 37°C.
• lOOmM L-lactate exerted a greater antimicrobial effect than lOOmM D-lactate on E. coli 0157 :H7 strain NCTC 12900.
• Increasing the proportion of the L-isomer over the D-isomer increased the antimicrobial efficacy in a dose- dependent manner for both strains .
• the antimicrobial effect of L-lactate and Coumarin on pathogens other than E. coli was examined.
• the strains examined were Salmonella enteri tidis NCTC 4444, Listeria monocytogenes NCTC 11994 and the methicillin-resistant Staphylococcus aureus (MRSA) strain NCTC 10442.
• Figure 8B illustrates the affect of Coumarin and L- lactate on L . monocytogenes .
• Coumarin (lOmM) alone had no effect on viability whereas L-lactate (50mM) reduced viability by greater than 5 logio units in 8 hours.
• L-lactate For certain applications it was desirable to determine potential synergy between L-lactate and Coumarin at the concentrations of these compounds likely to be used in commercial-like environments.
• the concentration of L-lactate was 2% and that of Coumarin 6.8mM.
• a range of 8 E. coli 0157 strains and 8 non-0157 E. coli strains were tested against these compounds and the viability of the strains determined at the start of the experiment and after 1 hour. Survival was calculated as previously.
• Coumarin alone had little effect on the survival of the E. coli strains whereas L-lactate reduced survival to between 0.00001% and 46%.
• L-lactate and Coumarin in combination reduced survival to between 0.00001% and 0.1%.
• NCTC 12900 was prepared as for the above experiments. The culture was then diluted into fresh media and incubated at 37°C for 2 hours. L-lactate and/or a coumarin were added and the cultures were re-incubated over a period of time. Bacterial growth was monitored by spectrophotometer (650nm) and compared to a control lacking both L- lactate and a coumarin.

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